Marine Sponge Metagenomics 17.3 Application and Latest Progress in Sponge Metagenomics 465
Part C | 17.3
cation for sequencing (Fig. 17.2). Cell singularization
mainly depends on the characteristics of the sample. If
possible, for pure cultures obtained from symbionts, serial dilution is a promising method. Commonly used
methods for cell singularization is fluorescence activated cell sorting (FACS). Target cells can be detected
by FISH or labeled with a fluorescent dye.
Microfluidic chambers have also proven to be
successful for obtaining single amplified genomes
(SAG) [17.53]. This method uses reaction volumes of
only 60 nl, which reduces the likelihood of contaminating the sample. Microfluidic devices have also been
developed recently for FISH and tyramide signal amplification FISH (tsa-FISH) followed by cell sorting
via flow cytometry directly on the device [17.54, 55].
This approach holds great promise for 16S rRNA genebased identification of single cells, while bearing low
risks of contamination. Micromanipulation techniques
are useful to target morphologically distinct microorganisms. Micromanipulation using microcapillaries has
been used successfully in combination with FISH to target the single cell [17.56]. The next step is cell lysis to
provide the genomic material for consequent multiple
displacement amplification (MDA).
MDA randomly amplifies primed template DNA
using the Phi29 polymerase enzyme in an isothermal reaction. DNA synthesized by Phi29 polymerase becomes
directly accessible for the next polymerase molecule,
thus resulting in continuous DNA amplification. The
next step is the whole genome sequencing of MDA
products. Various sequencing methods have been employed for whole genome sequencing.
Most studies have relied on shotgun or paired end
libraries using 454 pyrosequencing. However, mixture
of 454 and Illumina have been also used to enhance
the genome coverage. Combinations of 454 and Sanger
sequencing have also been used. The first single-cell
genome was obtained using Sanger, 454, and Illumina
techniques [17.57]. With the evolution of new sequencing techniques, novel approaches such as the PacBio or
IonTorrent techniques might facilitate obtaining better
genomic data from single cells.
17.3 Application and Latest Progress in Sponge Metagenomics
17.3.1 Diversity of Bacterial Symbionts
in Sponges
The use of culture-independent tools has greatly accelerated the understanding of the phylogeny of spongeassociated microbes. Underpinning this development
is the application of next generation sequencing technologies. A recent study using 454 amplicon pyrosequencing revealed extraordinary species richness in
Australia’s Great Barrier Reef sponges (Ianthella basta,
Ircina ramose, and R. odorabile) [17.58]. An enormous
bacterial diversity was demonstrated in those sponges.
This includes the dominant bacterial taxa Chloroflexi,
Acidobacteria, Actinobacteria, and Proteobacteria (Alpha, Delta, Gamma), which are the same phyla that featured in conventional 16S rRNA gene libraries [17.4].
Newly discovered phyla by 454 sequencing (e.g., Deferribacters, Tenericutes) were also present, but only in
low abundance [17.58].
Next generation 16S rRNA gene tag sequencing
suggests that the sponge microbiome may be classified into three main groups (species specific, variable,
and core) [17.59, 60]. Species specific microbes comprise 72% of the taxa detected in sponges, whereas
only 2% of the detected taxa correspond to the core.
The five Mediterranean sponges Aplysina aerophoba,
Aplysina cavernicola, Ircinia variabilis, Petrosia ficiformis, and Pseudocorticium jarrei share a core microbiome, which includes the phyla Acidobacteria,
Chloroflexi, Nitrospira, Poribacteria, and Proteobacteria. The phylum Chlamydiae and Lenthisphaerae
appears to be found only in association with A. cavernicola and I. variabilis, respectively [17.59]. The
core communities are represented as globally distributed microbes that are horizontally acquired from
the environment by a sponge, whereas the species
specific community could consist of microbes that
are vertically acquired from the progenitor [17.60].
Investigation of Cymbastela concentrica microbiome
helped to further elucidate possible genetic mechanisms involved in the establishment of species specific
microbiomes [17.61]. The authors sequenced about
190 623 shotgun sequences and 3545 16S rRNA gene
sequences. C. concentric contains the vast majority
of Gammaproteobacteria, Phyllobacteriaceae, Sphingomondales, Neisseriales, and Nitrospiraceae. Based
on 16S rRNA gene identification (97% identity cutoff),
only 34 different OTUs were common between C. concentrica and the surrounding seawater, which supported
the idea of selection for a specific microbiome, possibly
Part C | 17.3
cation for sequencing (Fig. 17.2). Cell singularization
mainly depends on the characteristics of the sample. If
possible, for pure cultures obtained from symbionts, serial dilution is a promising method. Commonly used
methods for cell singularization is fluorescence activated cell sorting (FACS). Target cells can be detected
by FISH or labeled with a fluorescent dye.
Microfluidic chambers have also proven to be
successful for obtaining single amplified genomes
(SAG) [17.53]. This method uses reaction volumes of
only 60 nl, which reduces the likelihood of contaminating the sample. Microfluidic devices have also been
developed recently for FISH and tyramide signal amplification FISH (tsa-FISH) followed by cell sorting
via flow cytometry directly on the device [17.54, 55].
This approach holds great promise for 16S rRNA genebased identification of single cells, while bearing low
risks of contamination. Micromanipulation techniques
are useful to target morphologically distinct microorganisms. Micromanipulation using microcapillaries has
been used successfully in combination with FISH to target the single cell [17.56]. The next step is cell lysis to
provide the genomic material for consequent multiple
displacement amplification (MDA).
MDA randomly amplifies primed template DNA
using the Phi29 polymerase enzyme in an isothermal reaction. DNA synthesized by Phi29 polymerase becomes
directly accessible for the next polymerase molecule,
thus resulting in continuous DNA amplification. The
next step is the whole genome sequencing of MDA
products. Various sequencing methods have been employed for whole genome sequencing.
Most studies have relied on shotgun or paired end
libraries using 454 pyrosequencing. However, mixture
of 454 and Illumina have been also used to enhance
the genome coverage. Combinations of 454 and Sanger
sequencing have also been used. The first single-cell
genome was obtained using Sanger, 454, and Illumina
techniques [17.57]. With the evolution of new sequencing techniques, novel approaches such as the PacBio or
IonTorrent techniques might facilitate obtaining better
genomic data from single cells.
17.3 Application and Latest Progress in Sponge Metagenomics
17.3.1 Diversity of Bacterial Symbionts
in Sponges
The use of culture-independent tools has greatly accelerated the understanding of the phylogeny of spongeassociated microbes. Underpinning this development
is the application of next generation sequencing technologies. A recent study using 454 amplicon pyrosequencing revealed extraordinary species richness in
Australia’s Great Barrier Reef sponges (Ianthella basta,
Ircina ramose, and R. odorabile) [17.58]. An enormous
bacterial diversity was demonstrated in those sponges.
This includes the dominant bacterial taxa Chloroflexi,
Acidobacteria, Actinobacteria, and Proteobacteria (Alpha, Delta, Gamma), which are the same phyla that featured in conventional 16S rRNA gene libraries [17.4].
Newly discovered phyla by 454 sequencing (e.g., Deferribacters, Tenericutes) were also present, but only in
low abundance [17.58].
Next generation 16S rRNA gene tag sequencing
suggests that the sponge microbiome may be classified into three main groups (species specific, variable,
and core) [17.59, 60]. Species specific microbes comprise 72% of the taxa detected in sponges, whereas
only 2% of the detected taxa correspond to the core.
The five Mediterranean sponges Aplysina aerophoba,
Aplysina cavernicola, Ircinia variabilis, Petrosia ficiformis, and Pseudocorticium jarrei share a core microbiome, which includes the phyla Acidobacteria,
Chloroflexi, Nitrospira, Poribacteria, and Proteobacteria. The phylum Chlamydiae and Lenthisphaerae
appears to be found only in association with A. cavernicola and I. variabilis, respectively [17.59]. The
core communities are represented as globally distributed microbes that are horizontally acquired from
the environment by a sponge, whereas the species
specific community could consist of microbes that
are vertically acquired from the progenitor [17.60].
Investigation of Cymbastela concentrica microbiome
helped to further elucidate possible genetic mechanisms involved in the establishment of species specific
microbiomes [17.61]. The authors sequenced about
190 623 shotgun sequences and 3545 16S rRNA gene
sequences. C. concentric contains the vast majority
of Gammaproteobacteria, Phyllobacteriaceae, Sphingomondales, Neisseriales, and Nitrospiraceae. Based
on 16S rRNA gene identification (97% identity cutoff),
only 34 different OTUs were common between C. concentrica and the surrounding seawater, which supported
the idea of selection for a specific microbiome, possibly